Effects of three biologically active substances on the function and morphology of mammalian spermatozoa were studied.Phospholipase A_2, of which activity has been detected in the seminal plasma of bull, goat and human, accelerated acrosome reaction of hamster spermatozoa. Addition of this enzyme to bull epididymal spermatozoa increased the rate of their entry into zona-free hamster egg. This effect was indefinite for bull ejaculated spermatozoa, suggesting that they had already received influence of enzyme from seminal plasma.Maitotoxin, a toxic and Ca^<2+>-dependent substance of marine origin, was used to examine whether it can induce the acrosome reaction as does Ca ionophore (A23187) in the mammalian spermatozoa. Maitotoxin inhibited motility and removed acrosome in the bull and goat spermatozoa when some 10 ng/ml were added. The less concentration (1-2ng/ml) induced almost normal acrodome reaction of spermatozoa and increased their entry into zona-free hamster egg. These results show that maitotoxin may be useful for inducer of acrosome reaction.Goat spermatozoa were used to extract cAMP-independent protein kinase and their substrate protein. Analyses of the purified material have shown that the protein kinase is similar to casein kinase II and there are peptides which are phosphorylated by this enzyme.